CD4 is the cellular receptor for HIV-1, the human retrovirus which causes the acquired immunodeficiency syndrome (AIDS). To elucidate the structure of CD4, we have exploited both eukaryotic and prokaryotic expression systems and produced a virus-binding fragment comprising domains 1-2 of the CD4 extracellular segment. A high resolution atomic structure has been solved at 2.4A and further refined to 2 angstroms. Site-directed mutagenesis in conjunction with kinetic and equilibrium binding studies identified the gp120 binding site as an about 900 angstroms2 patch on the C'C" ridge of the first Ig-like domain of CD4. Small organic inhibitors MW lesser than 1000) whose shape is predicted to be complementary to a surface groove near the virion binding site have been identified as leads for drug development using DOCK computational methods and their inhibitory activities verified in in vitro functional assays. The present proposal has three aims. First, we will produce and purify complexes of the 2 domain CD4 molecule (sCD41-183) with gp120. To this end, gp120 derived from HIV-1 SF2 or other strains will be expressed and complexed to sCD41-183. These complexes as well as partially deglycosylate complexes generated by N-glycanase and neuraminidase treatment of pre-formed complexes will be crystallized and structures solved by taking advantage of the known sCD41-183 coordinates for phasing and/or employing heavy atom derivatives. If no suitable crystals are obtained, complexes of sCD41-183 with CD4-binding glycosylation/truncation variants will be produced. The latter will be made using a baculovirus expression system and as a starting point, a construct of an envelope variant, gp59, previously shown to have native nanomolar affinity for CD4 even though it represents only 59% of the native gp120 amino acid sequence. Deletion of additional N-linked glycosylation sites will be predicted on electrospray-MS mapping studies which identify gp120 glycans protected by sCD41-183 from enzymatic deglycosylation. Once the key binding residues of several gp120 molecules are identified by x-ray analysis and confirmed in functional studies with point mutants of gp120, coordinates of the CD4 binding cavity on the surface of gp120 will provide a template for further design of complementary inhibitors. These compounds will serve as drug leads in project 6. Second, because it is imperative that anti-HIV compounds lack immunosuppressive activity, the class II MHC binding site on CD4 and any potential overlap with the HIV binding site will be fully characterized through use of a cellular CD4-class II MHC adhesion assay and informative mutagenesis studies. Third, constrained monocyclic and bicyclic peptides based upon 40Q-G-S-F43 and 37L-G-N-Q-G-S-F-L-T-K46 segments of the C'C" ridge of CD4 will be produced, characterized by solution NMR and tested for functional activity. Peptides which are structural mimics of the gp120 binding ridge and which selectively inhibits the CD4-HIV-1 gp120 interaction will be converted to peptidomimetics in Project 6.